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Sperm accumulation in the ampullae and cauda epididymides of bulls
Animal Reproduction Science 102 (2007) 238–246
Sperm accumulation in the ampullae and cauda epididymides of bulls Albert D. Barth ∗ Department of Large Animal Clinical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Sask. S7N 5B4, Canada Received 20 September 2006; accepted 8 November 2006 Available online 15 November 2006
Abstract Bulls that appear to have an abnormality of sperm transport accumulate large numbers of senescent sperm in the excurrent ducts of the reproductive tract. Six bulls that accumulated sperm were used to determine the number of physiological ejaculations required to deplete accumulated senescent sperm, semen traits during depletion, the period of time to re-accumulate senescent sperm after depletion, the sites of sperm accumulation, and the effect of sperm accumulation on fertility during natural service. Semen was collected from three bulls (HH, PH1, and CH1) three times daily using internal artificial vaginas placed in cows in estrus. These bulls started to produce semen with ≥70% morphologically normal sperm on the third, fifth, and seventh day, respectively. The percentage of live sperm increased from 5% to 68%, 5% to 63%, and 18% to 68% in HH, PH1, and CH1, respectively. Two weeks later the same bulls were electroejaculated every second day for five electroejaculations to deplete stores of senescent sperm. Each time, electroejaculation was continued until the semen produced had a dilute appearance. The three bulls re-accumulated senescent sperm after 1 month of sexual rest. After re-accumulation of senescent sperm, the total volumes of semen in both ampullae recovered at slaughter from HH, PH1, and CH1 were 5.0, 5.0, and 9.5 ml, respectively. The volumes of semen in ampullae recovered at slaughter from two control bulls (RA and CHC) were 1.7 and 1.9 ml, respectively. The number of sperm recovered from both cauda epididymides of HH, PH1, CH1, RA and CHC was 37.3 × 109 , 23.3 × 109 , 15.0 × 109 , 6.9 × 109 , and 7.4 × 109 , respectively. Bull CH1 and a fourth bull (LM) that also accumulated sperm, started to produce semen with ≥70% morphologically normal sperm, and ≥60% progressively motile sperm on the third electroejaculation after depletion of senescent sperm by repeated electroejaculations. Pregnancy rates achieved by two bulls that accumulated senescent sperm (CH2 and PH2) were less (P < 0.05) during the first week of a 21-day breeding period and there was a tendency for lesser pregnancy rates at the end of the breeding period when compared with two normal control bulls. The present study indicates that bulls that accumulate senescent sperm may achieve greater pregnancy rates approximately 1 week after beginning a period of frequent ejaculation. ∗
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A.D. Barth / Animal Reproduction Science 102 (2007) 238–246
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Re-accumulation of an increased percentage of senescent sperm would likely occur after 1 month of sexual rest. © 2006 Elsevier B.V. All rights reserved. Keywords: Bulls; Ampullae; Sperm accumulation; Detached heads
1. Introduction The selection of bulls that are capable of successfully breeding cows in a limited breeding season is essential to profitable cattle production. A condition that may reduce herd reproductive performance is the use of bulls that accumulate senescent sperm in the reproductive tract. This condition was reported in 1.1% of western Canadian range bulls (Barth and Oko, 1989) and appears to be similar to the phenomenon in stallions (Pickett and Voss, 1973). Bulls that accumulate senescent sperm are readily identified in routine bull breeding soundness evaluation (BSE). The semen characteristics of affected bulls have been previously reported (Barth and Oko, 1989; Barth, 1997). In these cases, when semen is collected by electroejaculation, 20–40 ml of concentrated semen can be obtained at one time. Most sperm are dead and often the percentage of detached heads is elevated presumably due to senescence. Frequent ejaculation results in a return to increased percentages of motile sperm and fewer numbers of detached heads. Usually sperm morphology is normal as the problem is not related to a disturbance of spermatogenesis, but rather, abnormal sperm transport within the excurrent duct of the reproductive tract. In bulls, daily sperm production and output is relatively consistent throughout the year with some seasonal limitations (Almquist et al., 1958; Amann et al., 1966). If sperm are continuously produced in the testes in a regular fashion, sperm must be evacuated from the male reproductive tract through ejaculation, spontaneous emission, micturition, resorption, or masturbation. Sperm are most likely lost at micturition in the stallion (Pickett et al., 1977). A large proportion of the daily sperm production in sexually rested rams (Lino et al., 1967) and rabbits (Wrobel, 1998) can be accounted for in the urine. The location of sperm accumulation in bulls has not been determined; however, in stallions it appears the site of sperm accumulation is the ampullae (Pickett and Voss, 1973). In normal bulls, the number of sperm in the ampullae is sufficient for at least one normal ejaculation after recent castration or vasectomy (Wrobel, 1998). The objectives of the present study were to characterize the semen traits of bulls with sperm accumulation, sites of sperm accumulation, number of physiological ejaculations and electroejaculations required to deplete the reproductive tract of senescent sperm, period of time during which senescent sperm may re-accumulate and effect of sperm accumulation on fertility during natural service. 2. Materials and methods Bulls (n = 6) that were diagnosed as sperm accumulators during routine breeding soundness examinations were used in four experiments. The bulls included one Horned Hereford (HH), two Polled Herefords (PH1 and PH2), two Charolais (CH1 and CH2), and one Limousin (LM). Bulls HH, PH1, CH1, PH2, CH2, and LM were aged 3, 3, 2, 4, 2, and 1 years, respectively. The scrotal circumference measurements of these bulls were 38, 37, 39, 37, 40 and 35 cm, respectively. The
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bulls were maintained on good quality brome-alfalfa hay at the Western College of Veterinary Medicine for periods of 6–18 months. Semen was evaluated according the method of the Western Canadian Association of Bovine Practitioners (WCABP) in warm laboratory conditions with warm microscope slides (Barth, 2000). Gross motility was evaluated at 40× magnification with bright field microscopy and individual progressive motility was evaluated with phase contrast microscopy at 400× magnification. Semen smears were stained with eosin–nigrosin for evaluation of sperm viability and morphology. Differential counts of sperm morphology were conducted using bright field microscopy at 1000× magnification. Sperm stained with eosin were considered to be dead while unstained sperm were considered to be alive. For purposes of the present study sperm accumulation was defined by the ability to ejaculate ≥20 ml of concentrated semen (estimated to be ≥750 millions sperm per ml) at one semen collection period in one day with ≥90% dead sperm as determined by microscopic observation of progressive motility and with eosin-nigrosin stained semen smears. Satisfactory semen quality was defined by the presence of ≥60% of progressively motile sperm and/or ≥60% unstained sperm, and ≥70% morphologically normal sperm. In the first experiment, HH, PH1, and CH1 were used to monitor changes in semen quality during sequential semen collections using internal artificial vaginas (IAV) placed in restrained cows (Barth et al., 2004). Bulls were allowed to serve cows with IAV three times daily until sperm viability returned to ≥60% of progressively motile sperm. At the same time, two control bulls, a Red Angus bull (RA) and a 2-year-old Charolais cross (CHC) with scrotal circumferences of 38 and 39 cm, respectively, that were producing semen of satisfactory quality, were allowed to serve cows with IAV once daily. If a control bull failed to serve a cow a semen sample was taken by electroejaculation. Two weeks after the first experiment, a second experiment was conducted to estimate the time that it takes for sperm accumulator bulls to produce satisfactory semen after repeated electroejaculations. HH, PH1, CH1 and LM were electroejaculated every second day until they began to produce semen with ≥60% progressively motile sperm, then they were given sexual rest to monitor the amount of time needed to re-accumulate ≥90% dead sperm. During sexual rest, the ampullae and the epididymides were examined weekly by ultrasonography, (Aloka SSD-900 scanner with a 5 MHz transducer, Aloka Co., Tokyo, Japan). Ultrasonographic images of the ampullae and epididymides were compared with images from two control bulls (CHC and RA). Two and 4 weeks after the onset of sexual rest 1 ml of semen was collected by electroejaculation to monitor increasing sperm senescence. Only 1 ml of semen was collected to avoid depleting the accumulating sperm. In a third experiment, CH2 and PH2 were used in single-sire pen-breeding trials to determine the effect of sperm accumulation on pregnancy rates after natural breeding. Two 3-year-old Polled Hereford bulls with satisfactory breeding potential (Barth, 2000) were used as controls. All bulls were examined for serving capacity (Barth, 2000) no more than 2 months prior to the breeding trials and were shown to have medium or high serving capacity. Before use in the trial, heifers were examined by rectal palpation and their reproductive tracts were scored from 1 to 5 for maturity (Andersen et al., 1991). Only heifers with score of 4 or 5 were selected for this study. Each bull was placed in a small pen with 30 heifers for 21 days. Breeding was observed for 2 h twice daily and all bulls were fitted with chin ball markers. Heifers that did not show signs of estrus, were not observed to be bred, and did not have paint marks on them, were removed from the experiment. These were also found to be non-pregnant. Pregnancy was determined by ultrasonography 27 days after the end of the breeding period.
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In a fourth experiment, three sexually rested sperm accumulator bulls HH, PH1, CH1 and two control bulls (CHC and RA) were slaughtered and the testes and accessory sex glands were recovered. The ampullae were flushed with known amounts of saline to determine the volume of semen and the number sperm stored in the ampullae. Epididymides were transected between the distal third and proximal two thirds of the cauda epididymis. The vas deferens was cannulated with a blunt 20 g needle and sperm were flushed from the distal third of the cauda epididymis with a known amount of saline. Sperm flushed from the epididymis were evaluated for morphology and the percent live sperm. Epididymal sperm reserve was determined for each cauda epididymis. The cauda epididymides were weighed prior to homogenization (Brinkmann/Kinematica POLYTRON® Benchtop Homogenizer, VWR International Inc., West Chester, PA, USA) with a known volume of saline. Sperm previously flushed from the distal one third of the cauda and the tissue homogenates were combined and mixed. Sperm concentration was determined by hemacytometer to allow calculation of the total number of sperm in each cauda epididymis. Pregnancy rates for two sperm accumulator bulls (CH2 and PH2) were compared with pregnancy rates of two control bulls by Chi-square analysis. 3. Results After three natural services per day using cows with IAVs, HH, PH1, and CH1 started to produce semen with >60% live sperm and >70% morphologically normal sperm on the third, fifth, and seventh day, respectively (Table 1). The percentage of live sperm increased from 5% to 68%, 5% to 63%, and 18% to 68% in HH, PH1, and CH1, respectively. The mean percentage of morphologically normal sperm increased from 27% to 73%, 32% to 76%, and 28% to 76% in Table 1 Number of services required to deplete senescent sperm from the excurrent ducts of three sperm accumulator bulls (HH, PH1, CH1) that served cows with inserted internal artificial vaginas 3× per day for up to 7 days Day 1
Day 2
Day 3
Day 4
Day 5
HH % detached heads % other sperm defects % morphologically normal % staining alive Volumea (ml)
62 13 27 5 4
36 13 54 21 3
17 11 73 68 2
PH1 % detached heads % other sperm defects % morphologically normal % staining alive Volumea (ml)
66 2 32 5 4
59 2 39 24 3
37 5 58 40 3
39 5 55 38 2
21 4 76 63 3
CH1 % detached heads % other sperm defects % morphologically normal % staining alive Volumea (ml)
68 4 28 18 3
52 10 38 32 4
42 11 49 45 5
41 3 56 45 4
32 1 67 70 4
Values are means rounded to nearest percentage or volume for three ejaculates on each day. a Some semen was not recoverable from the artificial vaginas.
Day 6
Day 7
19 15 67 71 5
18 5 76 68 4
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Fig. 1. Ultrasonograms of a cross-sectional view of the ampullae in a normal bull (A) and in a sperm accumulator bull (B). Arrows delimit dorsal and ventral borders of the ampullae.
HH, PH1, and CH1, respectively, due mainly to a decline in the percentage of detached sperm heads. The percentages of the other morphological sperm defects varied little during this period. HH, PH1, CH1 and LM, had re-accumulated senescent sperm (by definition used for a diagnosis of sperm accumulation) after 1 month of sexual rest following the depletion of senescent sperm by electroejaculation (Table 2). The percentage of detached heads after depletion by electroejaculation ranged from 12% to 18% and after 1 month of sexual rest ranged from 47% to 63% among these four bulls. After depletion by electroejaculation, progressive motility of sperm was ≥65%, but after 1 month of sexual rest had declined to ≤10%. During the first 2 weeks of sexual rest, there was no visible difference in the size and echotexture of ampullae in affected bulls and control bulls, but during the third and fourth weeks the luminal diameter of the ampullae of affected bulls began to increase (Fig. 1). In a longitudinal sectional view, the ampullae of affected bulls developed visibly larger anechoic fluid-filled lumens than those of the control bulls (Fig. 2). No differences could be detected in the ultrasonographic appearance of the cauda epididymides of affected bulls and control bulls. Data for results of the breeding trial using two bulls that accumulated sperm (CH2 and PH2) and two control bulls are shown in Table 3. Pregnancy rates during the first week of the breeding period were less (P < 0.05) for CH2 and PH2 when compared with control bulls. After the 21-day breeding period, pregnancy rates for CH2 and PH2 tended to be less than for the control bulls, but did not differ significantly (P > 0.05). The results indicate that it may take about 1 week of breeding for affected bulls to rid themselves of senescent sperm and then pregnancies would begin to occur at a normal rate.
Fig. 2. Ultrasonogram of a longitudinal sectional view of the ampullae in a normal bull (A) and in a sperm accumulator bull (B). Arrows delimit dorsal and ventral borders of the ampullae.
Bull
Volumea (ml) after EEJ
Volumea (ml) after 1 month
Detached heads (%) after EEJ
Detached heads (%) after 1 month
Normal sperm (%) after EEJ
Normal sperm (%) after 1 month
Motile sperm (%) after EEJ
Motile sperm (%) after 1 month
HH PH1 CH1 LM
4 4 4 5
39 40 56 35
14 12 18 15
60 47 54 63
73 83 76 78
29 48 36 30
65 76 65 80
5 5 5 10
a
Electroejaculation was continued until the concentration of semen emitted became dilute as in the appearance of skim milk.
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Table 2 Spermiograms of four sperm accumulator bulls that were electroejaculated (EEJ) every second day (3× over 6 days) to deplete stores of senescent sperm and then allowed 1 month of sexual rest for accumulation of senescent sperm
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Table 3 Pregnancy rates (%) for two sperm accumulator bulls (CH2 and PH2) and two Polled Hereford control bulls during a 21-day breeding period CH2
PH control 1
Week 1 Week 2 Week 3
16.7 (2/12) a 75.0 (6/8) 90.0 (9/10)
62.5 (5/8) b 100.0 (6/6) 75.0 (9/12)
Total
56.7 (17/30)
76.9 (20/26)
PH2
PH control 2
Week 1 Week 2 Week 3
0.0 (0/6) a 75.0 (6/8) 90.0 (9/10)
60.0 (3/5) b 80.0 (16/20) 50.0 (2/4)
Total
62.5 (15/24)
72.4 (21/29)
Examinations for pregnancy were done with ultrasonography 27 days after the end of the breeding period. The different letters indicate the pregnancy rates from the sperm accumulator bulls were significantly different (P < 0.05) from control bulls at the end of 1 week of the breeding period. Table 4 Characteristics of semen from the ampullae and cauda epididymides of three bulls that accumulated sperm (HH, PH1, CH1) and two control bulls (RA, CHC) Bull
Semen volume (ml) from both ampullae
Total sperm in both cauda epididymides (×106 )
Ampullae detached heads (%)
Cauda detached heads (%)
Ampullae live sperm (%)
Cauda live sperm (%)
HH PH1 CH1 RA CHC
5 5 9.5 1.7 1.9
37.3 23.4 15.0 6.9 7.4
38 52 72 4 3
3 6 3 3 2
24 52 13 95 95
79 88 93 94 97
After recovery of reproductive tracts at the slaughter of three affected bulls and two control bulls, the volumes of ampullary semen were larger in affected bulls. The total volumes of semen recovered from both ampullae of affected bulls ranged from 5.0 to 9.5 ml, whereas in two control bulls the volumes were 1.7 and 1.9 ml (Table 4). The total number of sperm recovered from both cauda epididymides from affected bulls ranged from 15 to 37.3 billion sperm compared to 6.9 and 7.4 billion sperm in the control bulls. The percentage of sperm with detached heads in the ampullae ranged from 38% to 72% in affected bulls as compared with 3% and 4% in two control bulls. However, there was little difference between affected and control bulls in the percentages of sperm with detached heads in the cauda epididymides. Somewhat similarly, the percentage of live sperm in the ampullae was less in affected bulls than in control bulls, but the percentage of live sperm in the cauda epididymides did not differ between affected and control bulls. 4. Discussion In bulls, as in other species, sperm are produced continuously, regardless of ejaculation frequency. Because sperm enter the epididymis at a constant rate, sperm must also leave the excurrent duct system at a relatively constant rate (Amman et al., 1974). The etiology of sperm accumulation in bulls is unknown; however, it could be postulated that sperm accumulator bulls have
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an abnormality in the sperm transport mechanism that results in accumulation of sperm in the epididymis and ampulla (Barth and Oko, 1989). Studies from other species demonstrate that contraction of the smooth muscle of the vas deferens has an important role in the propulsion of sperm into the pelvic urethra (Omu et al., 2001). There are several mechanisms involved in contractility of the epididymis and the vas deferens including factors altering electrical conductivity through the smooth muscle fibers of these structures, and hormonal influences such oxytocin and vasopressin that have been found to be present in the mammalian male reproductive tract and play a role in regulating contractility of the excurrent duct and probably sperm transport (Omu et al., 2001). Innervation of the excurrent duct system has a crucial role in sperm transport as well. It is possible that a disturbance of the sympathetic nerves emanating from the inferior mesenteric ganglion would result in sperm accumulation in some animals, because these nerves innervate the epididymis and vas deferens. In rats, removal of the inferior mesenteric ganglion resulted in excessive sperm accumulation of spermatozoa within the cauda epididymis (Ricker and Chang, 1996). Sperm accumulation in bulls has been associated with spinal disorders (Barth and Oko, 1989) and surgical vesiculectomy performed to resolve seminal vesiculitis (Nothling and Volkmann, 1997). In stallions, sperm accumulation was believed to be due to exhaustion of smooth muscle cells of the reproductive tract (Rasbech, 1975). Partial and complete blockages of the urethral openings of the ductus deferens and excretory ducts of vesicular glands have been reported in humans. These blockages were caused by inflammation or congenital abnormalities of the prostate (de Kretser, 2001). However, in the present study the prostate in sperm accumulator bulls was found to be grossly normal on post-slaughter dissection. One of the bulls in this study (LM) was a yearling and the author has observed other similarly affected yearling bulls as well as older bulls on previous occasions. Consequently, age does not appear to be a factor involved in the etiology of sperm accumulation in bulls. In the present study all bulls were able to breed cows normally, and none showed any physical abnormalities of any body system. No familial patterns for cases of sperm accumulation have been reported or observed by the author. Examination of the ampullae can be easily accomplished by transrectal ultrasonography (Weber et al., 1988; Ricker and Chang, 1996). The diameter of the lumen of the ampullae in the sperm accumulator bulls in the present study was numerically greater than in the control bulls and greater than reported previously in normal bulls (Weber et al., 1988). In the present study, frequent electroejaculation reduced the diameter of the ampullae in sperm accumulator bulls, decreased the percentage of detached heads, and increased the percentage of progressively motile sperm in the semen samples. At slaughter, sperm from the ampullae of bulls with sperm accumulation had a greater percentage of detached heads than sperm from the cauda epididymides. Therefore, it appears that increasing sperm head detachment occurs with increasing senescence of sperm accumulating in the ampullae of sperm accumulator bulls. Caution must be taken when sperm accumulator bulls are evaluated for breeding purposes since bulls producing semen with a greater percentage of detached heads have been associated with lesser fertility and some cases sterility (Cooper and Peet, 1984). The results of the present study indicate that pregnancy rates during the first week of the breeding period were significantly less and also tended to be less after 3-weeks of breeding for bulls that accumulated sperm than for control bulls. Thus, it would seem prudent that bulls identified with sperm accumulation at the time of BSE, should be classified as of questionable breeding soundness (Barth, 2000). These bulls might be given a satisfactory BSE status if the ampullary and epididymal sperm stores were depleted by frequent ejaculations near the beginning of a breeding season. Because re-accumulation occurs slowly over approximately 1 month it would seem likely that affected bulls would maintain a high percentage of viable sperm throughout the breeding season, providing that the breeding season
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was short and females to be bred were in normal estrous cyclicity. Sperm accumulation in bulls appears to be a permanent condition because two of the bulls in the present study were examined over two consecutive years without improvement. In conclusion, sperm accumulator bulls will store large amounts of senescent sperm in the ampullae and cauda epididymis, and frequent ejaculations over a period of about 1 week are needed for depletion of senescent sperm. Re-accumulation of senescent sperm occurs over approximately 1 month in sexually rested sperm accumulator bulls. Sperm accumulation appeared to be a permanent condition that will affect the pregnancy rates during the early period of breeding seasons. Acknowledgements The present study was funded by Saskatchewan Agriculture and Food. Thanks are given to clinical associate Andres Arteaga and farm manager Bill Kerr for their assistance in carrying out the project. References Almquist, J.O., Amann, R.P., O’Dell, W.T., 1958. Sperm reserves of dairy bulls as determined by depletion trials and post-slaughter sperm counts. J. Dairy Sci. 41, 733. Amann, R.P., Almquist, J.O., Lambiase, J.T., 1966. Seasonal cycles in bull semen characteristics. J. Anim. Sci. 25, 916. Amman, R.P., Kavanaugh, J.F., Griel, L.C., Voglmayr, J.K., 1974. Sperm production of Holstein bulls determined from testicular spermatid reserves, after cannulation of the rete testis or vas deferens, and by daily ejaculation. J. Dairy Sci. 57, 93–99. Andersen, K.J., LeFever, D.G., Brink, s.J.S., Odde, K.G., 1991. The use of reproductive tract scoring in beef heifers. Agri-Practice 12 (4), 19–26. Barth, A.D., Oko, R.J., 1989. Abnormal Morphology of Bovine Spermatozoa. Blackwell Publishing Inc., Malden, USA. Barth, A.D., 1997. Analysis of the bovine spermiogram. In: Proceedings of the Ann. Mtg. Soc. for Theriogenology, Montreal, pp. 123–132. Barth, A.D., Arteaga, A.A., Brito, L.F.C., Palmer, C.W., 2004. Use of internal artificial vaginas for breeding soundness evaluation in range bulls: an alternative for electroejaculation allowing observation on sex drive and mating ability. Anim. Reprod. Sci. 84, 315–325. Barth, A.D., 2000. Bull Breeding Soundness Evaluation. The Western Canadian Association of Bovine Practitioners, Saskatoon. Cooper, A.M., Peet, R.L., 1984. Infertility in a Hereford bull associated with increased number of detached heads in his ejaculated. Aust. Vet. J. 60, 225. de Kretser, D.M., 2001. Blockages of sperm transport. Androl. Aust. 321 (4), 499–503. Lino, B.F., Braden, A.W., Turnbull, K.E., 1967. Fate of the unejaculate spermatozoa. Nature 213, 594–595. Nothling, J.O., Volkmann, D.H., 1997. Dilatation of the ampullae and an increased incidence of loose sperm heads after bilateral vesiculectomy in a bull. Reprod. Domest. Anim. 32 (6), 321–324. Omu, A.E., Al-Bader, A.A., Dashti, H., Oriowo, M.A., 2001. Effect of extracellular Mg concentration on electrically induced contractions of rat vas deferent in vivo. Arch. Androl. 46 (3), 159–167. Pickett, B.W., Voss, J.L., 1973. Reproductive Management of the Stallion. General Series 934. Colorado State University. Experiment Station. Fort Collins. Pickett, B.W., Voss, J.L., Squirres, E.L., 1977. Impotence and abnormal sexual behavior in stallions. Theriogenology 8, 329–347. Rasbech, N.O., 1975. Ejaculatory disorders of the stallion. J. Reprod. Fertil. Suppl. 23, 123–128. Ricker, D.D., Chang, T.S.K., 1996. Neural input from the inferior mesenteric ganglion (IMG) affects sperm transport within the rat cauda epididymis. Int. J. Androl. 19, 371–376. Weber, J.A., Hilt, C.J., Woods, G.L., 1988. Ultrasonographic appearance of bull accessory sex glands. Theriogenology 20, 1347–1355. Wrobel, K.H., 1998. Male reproductive system. In: Dellmann, H.D., Eurell, J. (Eds.), Textbook of Veterinary Histology, 5th ed. Williams & Wilkins, p. 238.
Sperm accumulation in the ampullae and cauda epididymides of bulls Albert D. Barth ∗ Department of Large Animal Clinical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Sask. S7N 5B4, Canada Received 20 September 2006; accepted 8 November 2006 Available online 15 November 2006
Abstract Bulls that appear to have an abnormality of sperm transport accumulate large numbers of senescent sperm in the excurrent ducts of the reproductive tract. Six bulls that accumulated sperm were used to determine the number of physiological ejaculations required to deplete accumulated senescent sperm, semen traits during depletion, the period of time to re-accumulate senescent sperm after depletion, the sites of sperm accumulation, and the effect of sperm accumulation on fertility during natural service. Semen was collected from three bulls (HH, PH1, and CH1) three times daily using internal artificial vaginas placed in cows in estrus. These bulls started to produce semen with ≥70% morphologically normal sperm on the third, fifth, and seventh day, respectively. The percentage of live sperm increased from 5% to 68%, 5% to 63%, and 18% to 68% in HH, PH1, and CH1, respectively. Two weeks later the same bulls were electroejaculated every second day for five electroejaculations to deplete stores of senescent sperm. Each time, electroejaculation was continued until the semen produced had a dilute appearance. The three bulls re-accumulated senescent sperm after 1 month of sexual rest. After re-accumulation of senescent sperm, the total volumes of semen in both ampullae recovered at slaughter from HH, PH1, and CH1 were 5.0, 5.0, and 9.5 ml, respectively. The volumes of semen in ampullae recovered at slaughter from two control bulls (RA and CHC) were 1.7 and 1.9 ml, respectively. The number of sperm recovered from both cauda epididymides of HH, PH1, CH1, RA and CHC was 37.3 × 109 , 23.3 × 109 , 15.0 × 109 , 6.9 × 109 , and 7.4 × 109 , respectively. Bull CH1 and a fourth bull (LM) that also accumulated sperm, started to produce semen with ≥70% morphologically normal sperm, and ≥60% progressively motile sperm on the third electroejaculation after depletion of senescent sperm by repeated electroejaculations. Pregnancy rates achieved by two bulls that accumulated senescent sperm (CH2 and PH2) were less (P < 0.05) during the first week of a 21-day breeding period and there was a tendency for lesser pregnancy rates at the end of the breeding period when compared with two normal control bulls. The present study indicates that bulls that accumulate senescent sperm may achieve greater pregnancy rates approximately 1 week after beginning a period of frequent ejaculation. ∗
Tel.: +1 306 966 7151; fax: +1 306 966 7159. E-mail address: [email protected].
0378-4320/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2006.11.005
A.D. Barth / Animal Reproduction Science 102 (2007) 238–246
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Re-accumulation of an increased percentage of senescent sperm would likely occur after 1 month of sexual rest. © 2006 Elsevier B.V. All rights reserved. Keywords: Bulls; Ampullae; Sperm accumulation; Detached heads
1. Introduction The selection of bulls that are capable of successfully breeding cows in a limited breeding season is essential to profitable cattle production. A condition that may reduce herd reproductive performance is the use of bulls that accumulate senescent sperm in the reproductive tract. This condition was reported in 1.1% of western Canadian range bulls (Barth and Oko, 1989) and appears to be similar to the phenomenon in stallions (Pickett and Voss, 1973). Bulls that accumulate senescent sperm are readily identified in routine bull breeding soundness evaluation (BSE). The semen characteristics of affected bulls have been previously reported (Barth and Oko, 1989; Barth, 1997). In these cases, when semen is collected by electroejaculation, 20–40 ml of concentrated semen can be obtained at one time. Most sperm are dead and often the percentage of detached heads is elevated presumably due to senescence. Frequent ejaculation results in a return to increased percentages of motile sperm and fewer numbers of detached heads. Usually sperm morphology is normal as the problem is not related to a disturbance of spermatogenesis, but rather, abnormal sperm transport within the excurrent duct of the reproductive tract. In bulls, daily sperm production and output is relatively consistent throughout the year with some seasonal limitations (Almquist et al., 1958; Amann et al., 1966). If sperm are continuously produced in the testes in a regular fashion, sperm must be evacuated from the male reproductive tract through ejaculation, spontaneous emission, micturition, resorption, or masturbation. Sperm are most likely lost at micturition in the stallion (Pickett et al., 1977). A large proportion of the daily sperm production in sexually rested rams (Lino et al., 1967) and rabbits (Wrobel, 1998) can be accounted for in the urine. The location of sperm accumulation in bulls has not been determined; however, in stallions it appears the site of sperm accumulation is the ampullae (Pickett and Voss, 1973). In normal bulls, the number of sperm in the ampullae is sufficient for at least one normal ejaculation after recent castration or vasectomy (Wrobel, 1998). The objectives of the present study were to characterize the semen traits of bulls with sperm accumulation, sites of sperm accumulation, number of physiological ejaculations and electroejaculations required to deplete the reproductive tract of senescent sperm, period of time during which senescent sperm may re-accumulate and effect of sperm accumulation on fertility during natural service. 2. Materials and methods Bulls (n = 6) that were diagnosed as sperm accumulators during routine breeding soundness examinations were used in four experiments. The bulls included one Horned Hereford (HH), two Polled Herefords (PH1 and PH2), two Charolais (CH1 and CH2), and one Limousin (LM). Bulls HH, PH1, CH1, PH2, CH2, and LM were aged 3, 3, 2, 4, 2, and 1 years, respectively. The scrotal circumference measurements of these bulls were 38, 37, 39, 37, 40 and 35 cm, respectively. The
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bulls were maintained on good quality brome-alfalfa hay at the Western College of Veterinary Medicine for periods of 6–18 months. Semen was evaluated according the method of the Western Canadian Association of Bovine Practitioners (WCABP) in warm laboratory conditions with warm microscope slides (Barth, 2000). Gross motility was evaluated at 40× magnification with bright field microscopy and individual progressive motility was evaluated with phase contrast microscopy at 400× magnification. Semen smears were stained with eosin–nigrosin for evaluation of sperm viability and morphology. Differential counts of sperm morphology were conducted using bright field microscopy at 1000× magnification. Sperm stained with eosin were considered to be dead while unstained sperm were considered to be alive. For purposes of the present study sperm accumulation was defined by the ability to ejaculate ≥20 ml of concentrated semen (estimated to be ≥750 millions sperm per ml) at one semen collection period in one day with ≥90% dead sperm as determined by microscopic observation of progressive motility and with eosin-nigrosin stained semen smears. Satisfactory semen quality was defined by the presence of ≥60% of progressively motile sperm and/or ≥60% unstained sperm, and ≥70% morphologically normal sperm. In the first experiment, HH, PH1, and CH1 were used to monitor changes in semen quality during sequential semen collections using internal artificial vaginas (IAV) placed in restrained cows (Barth et al., 2004). Bulls were allowed to serve cows with IAV three times daily until sperm viability returned to ≥60% of progressively motile sperm. At the same time, two control bulls, a Red Angus bull (RA) and a 2-year-old Charolais cross (CHC) with scrotal circumferences of 38 and 39 cm, respectively, that were producing semen of satisfactory quality, were allowed to serve cows with IAV once daily. If a control bull failed to serve a cow a semen sample was taken by electroejaculation. Two weeks after the first experiment, a second experiment was conducted to estimate the time that it takes for sperm accumulator bulls to produce satisfactory semen after repeated electroejaculations. HH, PH1, CH1 and LM were electroejaculated every second day until they began to produce semen with ≥60% progressively motile sperm, then they were given sexual rest to monitor the amount of time needed to re-accumulate ≥90% dead sperm. During sexual rest, the ampullae and the epididymides were examined weekly by ultrasonography, (Aloka SSD-900 scanner with a 5 MHz transducer, Aloka Co., Tokyo, Japan). Ultrasonographic images of the ampullae and epididymides were compared with images from two control bulls (CHC and RA). Two and 4 weeks after the onset of sexual rest 1 ml of semen was collected by electroejaculation to monitor increasing sperm senescence. Only 1 ml of semen was collected to avoid depleting the accumulating sperm. In a third experiment, CH2 and PH2 were used in single-sire pen-breeding trials to determine the effect of sperm accumulation on pregnancy rates after natural breeding. Two 3-year-old Polled Hereford bulls with satisfactory breeding potential (Barth, 2000) were used as controls. All bulls were examined for serving capacity (Barth, 2000) no more than 2 months prior to the breeding trials and were shown to have medium or high serving capacity. Before use in the trial, heifers were examined by rectal palpation and their reproductive tracts were scored from 1 to 5 for maturity (Andersen et al., 1991). Only heifers with score of 4 or 5 were selected for this study. Each bull was placed in a small pen with 30 heifers for 21 days. Breeding was observed for 2 h twice daily and all bulls were fitted with chin ball markers. Heifers that did not show signs of estrus, were not observed to be bred, and did not have paint marks on them, were removed from the experiment. These were also found to be non-pregnant. Pregnancy was determined by ultrasonography 27 days after the end of the breeding period.
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In a fourth experiment, three sexually rested sperm accumulator bulls HH, PH1, CH1 and two control bulls (CHC and RA) were slaughtered and the testes and accessory sex glands were recovered. The ampullae were flushed with known amounts of saline to determine the volume of semen and the number sperm stored in the ampullae. Epididymides were transected between the distal third and proximal two thirds of the cauda epididymis. The vas deferens was cannulated with a blunt 20 g needle and sperm were flushed from the distal third of the cauda epididymis with a known amount of saline. Sperm flushed from the epididymis were evaluated for morphology and the percent live sperm. Epididymal sperm reserve was determined for each cauda epididymis. The cauda epididymides were weighed prior to homogenization (Brinkmann/Kinematica POLYTRON® Benchtop Homogenizer, VWR International Inc., West Chester, PA, USA) with a known volume of saline. Sperm previously flushed from the distal one third of the cauda and the tissue homogenates were combined and mixed. Sperm concentration was determined by hemacytometer to allow calculation of the total number of sperm in each cauda epididymis. Pregnancy rates for two sperm accumulator bulls (CH2 and PH2) were compared with pregnancy rates of two control bulls by Chi-square analysis. 3. Results After three natural services per day using cows with IAVs, HH, PH1, and CH1 started to produce semen with >60% live sperm and >70% morphologically normal sperm on the third, fifth, and seventh day, respectively (Table 1). The percentage of live sperm increased from 5% to 68%, 5% to 63%, and 18% to 68% in HH, PH1, and CH1, respectively. The mean percentage of morphologically normal sperm increased from 27% to 73%, 32% to 76%, and 28% to 76% in Table 1 Number of services required to deplete senescent sperm from the excurrent ducts of three sperm accumulator bulls (HH, PH1, CH1) that served cows with inserted internal artificial vaginas 3× per day for up to 7 days Day 1
Day 2
Day 3
Day 4
Day 5
HH % detached heads % other sperm defects % morphologically normal % staining alive Volumea (ml)
62 13 27 5 4
36 13 54 21 3
17 11 73 68 2
PH1 % detached heads % other sperm defects % morphologically normal % staining alive Volumea (ml)
66 2 32 5 4
59 2 39 24 3
37 5 58 40 3
39 5 55 38 2
21 4 76 63 3
CH1 % detached heads % other sperm defects % morphologically normal % staining alive Volumea (ml)
68 4 28 18 3
52 10 38 32 4
42 11 49 45 5
41 3 56 45 4
32 1 67 70 4
Values are means rounded to nearest percentage or volume for three ejaculates on each day. a Some semen was not recoverable from the artificial vaginas.
Day 6
Day 7
19 15 67 71 5
18 5 76 68 4
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Fig. 1. Ultrasonograms of a cross-sectional view of the ampullae in a normal bull (A) and in a sperm accumulator bull (B). Arrows delimit dorsal and ventral borders of the ampullae.
HH, PH1, and CH1, respectively, due mainly to a decline in the percentage of detached sperm heads. The percentages of the other morphological sperm defects varied little during this period. HH, PH1, CH1 and LM, had re-accumulated senescent sperm (by definition used for a diagnosis of sperm accumulation) after 1 month of sexual rest following the depletion of senescent sperm by electroejaculation (Table 2). The percentage of detached heads after depletion by electroejaculation ranged from 12% to 18% and after 1 month of sexual rest ranged from 47% to 63% among these four bulls. After depletion by electroejaculation, progressive motility of sperm was ≥65%, but after 1 month of sexual rest had declined to ≤10%. During the first 2 weeks of sexual rest, there was no visible difference in the size and echotexture of ampullae in affected bulls and control bulls, but during the third and fourth weeks the luminal diameter of the ampullae of affected bulls began to increase (Fig. 1). In a longitudinal sectional view, the ampullae of affected bulls developed visibly larger anechoic fluid-filled lumens than those of the control bulls (Fig. 2). No differences could be detected in the ultrasonographic appearance of the cauda epididymides of affected bulls and control bulls. Data for results of the breeding trial using two bulls that accumulated sperm (CH2 and PH2) and two control bulls are shown in Table 3. Pregnancy rates during the first week of the breeding period were less (P < 0.05) for CH2 and PH2 when compared with control bulls. After the 21-day breeding period, pregnancy rates for CH2 and PH2 tended to be less than for the control bulls, but did not differ significantly (P > 0.05). The results indicate that it may take about 1 week of breeding for affected bulls to rid themselves of senescent sperm and then pregnancies would begin to occur at a normal rate.
Fig. 2. Ultrasonogram of a longitudinal sectional view of the ampullae in a normal bull (A) and in a sperm accumulator bull (B). Arrows delimit dorsal and ventral borders of the ampullae.
Bull
Volumea (ml) after EEJ
Volumea (ml) after 1 month
Detached heads (%) after EEJ
Detached heads (%) after 1 month
Normal sperm (%) after EEJ
Normal sperm (%) after 1 month
Motile sperm (%) after EEJ
Motile sperm (%) after 1 month
HH PH1 CH1 LM
4 4 4 5
39 40 56 35
14 12 18 15
60 47 54 63
73 83 76 78
29 48 36 30
65 76 65 80
5 5 5 10
a
Electroejaculation was continued until the concentration of semen emitted became dilute as in the appearance of skim milk.
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Table 2 Spermiograms of four sperm accumulator bulls that were electroejaculated (EEJ) every second day (3× over 6 days) to deplete stores of senescent sperm and then allowed 1 month of sexual rest for accumulation of senescent sperm
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Table 3 Pregnancy rates (%) for two sperm accumulator bulls (CH2 and PH2) and two Polled Hereford control bulls during a 21-day breeding period CH2
PH control 1
Week 1 Week 2 Week 3
16.7 (2/12) a 75.0 (6/8) 90.0 (9/10)
62.5 (5/8) b 100.0 (6/6) 75.0 (9/12)
Total
56.7 (17/30)
76.9 (20/26)
PH2
PH control 2
Week 1 Week 2 Week 3
0.0 (0/6) a 75.0 (6/8) 90.0 (9/10)
60.0 (3/5) b 80.0 (16/20) 50.0 (2/4)
Total
62.5 (15/24)
72.4 (21/29)
Examinations for pregnancy were done with ultrasonography 27 days after the end of the breeding period. The different letters indicate the pregnancy rates from the sperm accumulator bulls were significantly different (P < 0.05) from control bulls at the end of 1 week of the breeding period. Table 4 Characteristics of semen from the ampullae and cauda epididymides of three bulls that accumulated sperm (HH, PH1, CH1) and two control bulls (RA, CHC) Bull
Semen volume (ml) from both ampullae
Total sperm in both cauda epididymides (×106 )
Ampullae detached heads (%)
Cauda detached heads (%)
Ampullae live sperm (%)
Cauda live sperm (%)
HH PH1 CH1 RA CHC
5 5 9.5 1.7 1.9
37.3 23.4 15.0 6.9 7.4
38 52 72 4 3
3 6 3 3 2
24 52 13 95 95
79 88 93 94 97
After recovery of reproductive tracts at the slaughter of three affected bulls and two control bulls, the volumes of ampullary semen were larger in affected bulls. The total volumes of semen recovered from both ampullae of affected bulls ranged from 5.0 to 9.5 ml, whereas in two control bulls the volumes were 1.7 and 1.9 ml (Table 4). The total number of sperm recovered from both cauda epididymides from affected bulls ranged from 15 to 37.3 billion sperm compared to 6.9 and 7.4 billion sperm in the control bulls. The percentage of sperm with detached heads in the ampullae ranged from 38% to 72% in affected bulls as compared with 3% and 4% in two control bulls. However, there was little difference between affected and control bulls in the percentages of sperm with detached heads in the cauda epididymides. Somewhat similarly, the percentage of live sperm in the ampullae was less in affected bulls than in control bulls, but the percentage of live sperm in the cauda epididymides did not differ between affected and control bulls. 4. Discussion In bulls, as in other species, sperm are produced continuously, regardless of ejaculation frequency. Because sperm enter the epididymis at a constant rate, sperm must also leave the excurrent duct system at a relatively constant rate (Amman et al., 1974). The etiology of sperm accumulation in bulls is unknown; however, it could be postulated that sperm accumulator bulls have
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an abnormality in the sperm transport mechanism that results in accumulation of sperm in the epididymis and ampulla (Barth and Oko, 1989). Studies from other species demonstrate that contraction of the smooth muscle of the vas deferens has an important role in the propulsion of sperm into the pelvic urethra (Omu et al., 2001). There are several mechanisms involved in contractility of the epididymis and the vas deferens including factors altering electrical conductivity through the smooth muscle fibers of these structures, and hormonal influences such oxytocin and vasopressin that have been found to be present in the mammalian male reproductive tract and play a role in regulating contractility of the excurrent duct and probably sperm transport (Omu et al., 2001). Innervation of the excurrent duct system has a crucial role in sperm transport as well. It is possible that a disturbance of the sympathetic nerves emanating from the inferior mesenteric ganglion would result in sperm accumulation in some animals, because these nerves innervate the epididymis and vas deferens. In rats, removal of the inferior mesenteric ganglion resulted in excessive sperm accumulation of spermatozoa within the cauda epididymis (Ricker and Chang, 1996). Sperm accumulation in bulls has been associated with spinal disorders (Barth and Oko, 1989) and surgical vesiculectomy performed to resolve seminal vesiculitis (Nothling and Volkmann, 1997). In stallions, sperm accumulation was believed to be due to exhaustion of smooth muscle cells of the reproductive tract (Rasbech, 1975). Partial and complete blockages of the urethral openings of the ductus deferens and excretory ducts of vesicular glands have been reported in humans. These blockages were caused by inflammation or congenital abnormalities of the prostate (de Kretser, 2001). However, in the present study the prostate in sperm accumulator bulls was found to be grossly normal on post-slaughter dissection. One of the bulls in this study (LM) was a yearling and the author has observed other similarly affected yearling bulls as well as older bulls on previous occasions. Consequently, age does not appear to be a factor involved in the etiology of sperm accumulation in bulls. In the present study all bulls were able to breed cows normally, and none showed any physical abnormalities of any body system. No familial patterns for cases of sperm accumulation have been reported or observed by the author. Examination of the ampullae can be easily accomplished by transrectal ultrasonography (Weber et al., 1988; Ricker and Chang, 1996). The diameter of the lumen of the ampullae in the sperm accumulator bulls in the present study was numerically greater than in the control bulls and greater than reported previously in normal bulls (Weber et al., 1988). In the present study, frequent electroejaculation reduced the diameter of the ampullae in sperm accumulator bulls, decreased the percentage of detached heads, and increased the percentage of progressively motile sperm in the semen samples. At slaughter, sperm from the ampullae of bulls with sperm accumulation had a greater percentage of detached heads than sperm from the cauda epididymides. Therefore, it appears that increasing sperm head detachment occurs with increasing senescence of sperm accumulating in the ampullae of sperm accumulator bulls. Caution must be taken when sperm accumulator bulls are evaluated for breeding purposes since bulls producing semen with a greater percentage of detached heads have been associated with lesser fertility and some cases sterility (Cooper and Peet, 1984). The results of the present study indicate that pregnancy rates during the first week of the breeding period were significantly less and also tended to be less after 3-weeks of breeding for bulls that accumulated sperm than for control bulls. Thus, it would seem prudent that bulls identified with sperm accumulation at the time of BSE, should be classified as of questionable breeding soundness (Barth, 2000). These bulls might be given a satisfactory BSE status if the ampullary and epididymal sperm stores were depleted by frequent ejaculations near the beginning of a breeding season. Because re-accumulation occurs slowly over approximately 1 month it would seem likely that affected bulls would maintain a high percentage of viable sperm throughout the breeding season, providing that the breeding season
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was short and females to be bred were in normal estrous cyclicity. Sperm accumulation in bulls appears to be a permanent condition because two of the bulls in the present study were examined over two consecutive years without improvement. In conclusion, sperm accumulator bulls will store large amounts of senescent sperm in the ampullae and cauda epididymis, and frequent ejaculations over a period of about 1 week are needed for depletion of senescent sperm. Re-accumulation of senescent sperm occurs over approximately 1 month in sexually rested sperm accumulator bulls. Sperm accumulation appeared to be a permanent condition that will affect the pregnancy rates during the early period of breeding seasons. Acknowledgements The present study was funded by Saskatchewan Agriculture and Food. Thanks are given to clinical associate Andres Arteaga and farm manager Bill Kerr for their assistance in carrying out the project. References Almquist, J.O., Amann, R.P., O’Dell, W.T., 1958. Sperm reserves of dairy bulls as determined by depletion trials and post-slaughter sperm counts. J. Dairy Sci. 41, 733. Amann, R.P., Almquist, J.O., Lambiase, J.T., 1966. Seasonal cycles in bull semen characteristics. J. Anim. Sci. 25, 916. Amman, R.P., Kavanaugh, J.F., Griel, L.C., Voglmayr, J.K., 1974. Sperm production of Holstein bulls determined from testicular spermatid reserves, after cannulation of the rete testis or vas deferens, and by daily ejaculation. J. Dairy Sci. 57, 93–99. Andersen, K.J., LeFever, D.G., Brink, s.J.S., Odde, K.G., 1991. The use of reproductive tract scoring in beef heifers. Agri-Practice 12 (4), 19–26. Barth, A.D., Oko, R.J., 1989. Abnormal Morphology of Bovine Spermatozoa. Blackwell Publishing Inc., Malden, USA. Barth, A.D., 1997. Analysis of the bovine spermiogram. In: Proceedings of the Ann. Mtg. Soc. for Theriogenology, Montreal, pp. 123–132. Barth, A.D., Arteaga, A.A., Brito, L.F.C., Palmer, C.W., 2004. Use of internal artificial vaginas for breeding soundness evaluation in range bulls: an alternative for electroejaculation allowing observation on sex drive and mating ability. Anim. Reprod. Sci. 84, 315–325. Barth, A.D., 2000. Bull Breeding Soundness Evaluation. The Western Canadian Association of Bovine Practitioners, Saskatoon. Cooper, A.M., Peet, R.L., 1984. Infertility in a Hereford bull associated with increased number of detached heads in his ejaculated. Aust. Vet. J. 60, 225. de Kretser, D.M., 2001. Blockages of sperm transport. Androl. Aust. 321 (4), 499–503. Lino, B.F., Braden, A.W., Turnbull, K.E., 1967. Fate of the unejaculate spermatozoa. Nature 213, 594–595. Nothling, J.O., Volkmann, D.H., 1997. Dilatation of the ampullae and an increased incidence of loose sperm heads after bilateral vesiculectomy in a bull. Reprod. Domest. Anim. 32 (6), 321–324. Omu, A.E., Al-Bader, A.A., Dashti, H., Oriowo, M.A., 2001. Effect of extracellular Mg concentration on electrically induced contractions of rat vas deferent in vivo. Arch. Androl. 46 (3), 159–167. Pickett, B.W., Voss, J.L., 1973. Reproductive Management of the Stallion. General Series 934. Colorado State University. Experiment Station. Fort Collins. Pickett, B.W., Voss, J.L., Squirres, E.L., 1977. Impotence and abnormal sexual behavior in stallions. Theriogenology 8, 329–347. Rasbech, N.O., 1975. Ejaculatory disorders of the stallion. J. Reprod. Fertil. Suppl. 23, 123–128. Ricker, D.D., Chang, T.S.K., 1996. Neural input from the inferior mesenteric ganglion (IMG) affects sperm transport within the rat cauda epididymis. Int. J. Androl. 19, 371–376. Weber, J.A., Hilt, C.J., Woods, G.L., 1988. Ultrasonographic appearance of bull accessory sex glands. Theriogenology 20, 1347–1355. Wrobel, K.H., 1998. Male reproductive system. In: Dellmann, H.D., Eurell, J. (Eds.), Textbook of Veterinary Histology, 5th ed. Williams & Wilkins, p. 238.